Genetic Studies Of MEI1 And SMYD5 Genes Associated With Early Embryonic Arrest In Human

Early embryonic arrest(EEA)is a multifactorial pathological condition characterized by delayed embryonic development,infertility due to failure to form a viable eight-cell embryo on day 3 of the assisted reproductive cycle.With the widespread application of whole exome sequencing technology,more and more genes are associated with EEA.At present,a number of pathogenic genes related to early embryonic development arrest in humans have been discovered,but only some cases can be explained,and there are still many patients with early embryonic development arrest whose etiology is unknown.The purpose of this study was to further explore genetic factors associated with EEA by expanding the patient cohort.Part Ⅰ Mutation Analysis of MEI1 in Infertile Females with Early Embryonic Developmental ArrestObjective:The present study was intended to screen novel variants in the known pathogenic genes from primary infertility patients with recurrent early embryonic developmental arrest in IVF/ICSI cycles.Methods:We recruited female patients with primary infertility characterized by recurrent early embryonic developmental arrest.Collected peripheral blood from the affected women and their families for DNA extraction.The currently known pathogenic genes in patients with early embryonic arrest were compared and new mutations in the known pathogenic genes were screened through whole-exome sequencing and data analysis.The candidate variants were validated via Sanger sequencing.By constructing wild-type and mutant plasmids in vitro,transfecting 293T cells,Western Blot detected their protein expression levels and detected the effects of MEI1(meiotic double-strand break formation protein 1)gene mutations on their expression and protein function.Results:We found the three novel mutations,(exon 12 deletion),c.122C>T(p.P41L)and c.3541C>T(p.R1181W)and a previously reported mutation c.1585T>A(p.F529I)in MEI1 in four patients from three independent families through whole-exome sequencing.These biallelic mutations can lead to early embryonic developmental arrest and female infertility.Immunofluorescence showed homozygous exon 12 deletion of MEI1 gene(Exon 12 deletion)causes spindle and chromosomal arrangement disorders.At the same time,western blot results suggest that mutations can lead to the expression of MEI1 protein.Conclusion:Our study extends the mutational and phenotypic spectrum of female infertility caused by mutations in MEI1 and further confirms previously reported findings that MEI1 plays an essential role in early embryonic development.Part Ⅱ Mutation Analysis of SMYD5 in Infertile Females with Early Embryonic Developmental ArrestObjective:This study aimed to screen for novel causative genes from patients with recurrent early embryonic developmental arrest unexplained by known causative genes.Methods:We recruited female patients with primary infertility characterized by recurrent early embryonic developmental arrest.Collected peripheral blood from the affected women and their families for DNA extraction.Whole-exome sequencing were performed to screen candidate mutations.We focused on harmful and rare genes that are highly expressed or specifically expressed in the ovary in cases where the known causative genes cannot be explained,so as to discover new candidate pathogenic genes.The candidate mutations were validated via Sanger sequencing.It was detected to be expressed in the ovaries and embryos at all levels,and the protein structure prediction results showed that the SMYD5 gene mutation led to changes in the protein structure.It is speculated that the mutation may affect the interaction between SMYD5 and related binding proteins,resulting in early embryonic development arrest.Results:Through whole exome sequencing,we found homozygous missense mutations of the SMYD5(c.832C>T:p.R278C)in a closely related family patient,and the peripheral blood sanger sequencing of the patient and parents was verified,and the family line was analyzed in line with the recessive inheritance pattern.A variety of biological software predictions and database analysis showed that the mutation frequency was very low in the normal population,and functional prediction found that the mutation sites were all harmful mutations.The variation found in this study is the first reported mutation of SMYD5 in early human embryonic developmental arrest.According to ACMG guidelines,the mutation is judged to be a suspected causative variant.The protein structure prediction results showed that SMYD5 gene mutations led to changes in protein structure.It is speculated that the mutation may affect the interaction between SMYD5 and related binding proteins,resulting in early embryonic development arrest.Conclusion:Our study found that the homozygous mutation c.832C>T in SMYD5 was associated with human early embryonic developmental arrest,which helps us understand the role of SMYD5 in human early embryonic development and provides a novel genetic marker for female infertility.